Supplemental Figure S1. (A) Schematic diagrams of CD20-specific CAR constructs. (B) Representative histogram of CAR expression. Supplemental Figure S2. (A) Gating strategy for analyzing proliferation of CFSE-stained T cells. (B) Histograms of CFSE dilution of CD3+ T cells corresponding to the experiment in Figure 2. Supplemental Figure S3. Proliferation and cytokine secretion of 1F5-28-BB-z CAR. Supplemental Figure S4. Proliferation, cytokine secretion, and cytotoxicity of CAR T cells with fully human anti-CD20 scFv. Supplemental Figure S5. CD20 expression of K80-20low, K80-20med, K80-20high as determined by flow cytometry. Supplemental Figure S6. The absolute cytokine concentrations from T cell supernatants from the experiment in Figure 4 are shown. Supplemental Figure S7. Rituximab-refractory Raji-ffLuc have the same CD20 expression as parental Raji-ffLuc cells. Supplemental Figure S8. (A) Individual mouse bioluminescent tumor burden traces over time corresponding to Figure 5. (B) Representative mouse bioluminescence images. Supplemental Figure S9. Presence of circulating T cells in mice. Supplemental Figure S10. Ofatumumab blocks antigen binding of Ab used to derive CAR scFv.
Funding
Damon Runyon-Pfizer Clinical Investigator
Giuliani Family Foundation
NIH/NCI
NIH/NCI Cancer Center Support
NIDDK
Lymphoma Research Foundation
ARTICLE ABSTRACT
CD20 is an attractive immunotherapy target for B-cell non-Hodgkin lymphomas, and adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) targeting CD20 is a promising strategy. A theoretical limitation is that residual serum rituximab might block CAR binding to CD20 and thereby impede T cell–mediated anti-lymphoma responses. The activity of CD20 CAR-modified T cells in the presence of various concentrations of rituximab was tested in vitro and in vivo. CAR-binding sites on CD20+ tumor cells were blocked by rituximab in a dose-dependent fashion, although at 37°C blockade was incomplete at concentrations up to 200 μg/mL. T cells with CD20 CARs also exhibited modest dose-dependent reductions in cytokine secretion and cytotoxicity, but not proliferation, against lymphoma cell lines. At rituximab concentrations of 100 μg/mL, CAR T cells retained ≥50% of baseline activity against targets with high CD20 expression, but were more strongly inhibited when target cells expressed low CD20. In a murine xenograft model using a rituximab-refractory lymphoma cell line, rituximab did not impair CAR T-cell activity, and tumors were eradicated in >85% of mice. Clinical residual rituximab serum concentrations were measured in 103 lymphoma patients after rituximab therapy, with the median level found to be only 38 μg/mL (interquartile range, 19–72 μg/mL). Thus, despite modest functional impairment in vitro, the in vivo activity of CD20-targeted CAR T cells remains intact at clinically relevant levels of rituximab, making use of these T cells clinically feasible. Cancer Immunol Res; 4(6); 509–19. ©2016 AACR.See related Spotlight by Sadelain, p. 473.